System and method for removal of immune inhibitors from biological fluids

ABSTRACT

The present system and method are useful for the removal of immune inhibitors such as soluble TNF receptors from the body fluid of cancer patients. In some embodiments, soluble TNF-Receptors 1 and 2 are selectively removed from plasma at 80% or more efficiency. In some embodiments, the system includes an immobilized capture ligand of a single chain TNFα. The system and method are useful for the treatment of different cancer types, stages and severity.

1. FIELD

The present disclosure pertains to a system and method for removal ofimmune inhibitors from plasma.

2. INTRODUCTION

Leveraging of the immune system to kill cancer has been the focus ofoncologists and cancer researchers for more than a century. Observationsthat patient tumors enter remission subsequent to immune stimulatingbacterial infections (see, for example, Coley, W. B. (1991). Clin OrthopRelat Res, 3-11; Hughes, W. T., and Smith, D. R. (1973) Cancer 31,1008-1014; Yates, J. W., and Holland, J. F. (1973) Cancer 32, 1490-1498;Muller, H. E. (1974) (author's translation), Pathol Microbiol (Basel)40, 297-304) as well as correlations between immune cell infiltration ofcancer and survival (see, for example, Lipponen, P. K., et al. (1992)Eur J Cancer 29A, 69-75; Ma, D., and Gu, M. J. (1991) J Tongji Med Univ11, 235-239; Pastrnak, A., and Jansa, P. (1989) Acta Univ Palacki OlomucFac Med 124, 7-71; Di Giorgio, et al. (1992) Int Surg 77, 256-260); DiGiorgio, A., et al. (1992) Int Surg 77, 256-260), have suggested thepossibility of immunological control of neoplasia. It was however, onlyin the last decade that workers in the field of cancer immunotherapyhave been able to claim significant improvements in patients' prognosis.A major accomplishment in the field was the development of antibodiesthat suppress the negative regulators or checkpoints of T-cellactivation. These antibodies belong to a class of drugs termed “immunecheckpoint inhibitors”. The first one cleared by the FDA, Ipilimumab, anantagonistic antibody targeting cytotoxic T-lymphocyte-associatedprotein 4 (CTLA-4), improved overall survival in metastatic melanomapatients in 2010. An associated study assessed a total of 676HLA-A*0201-positive patients with unresectable stage III or IV melanomawhich were assigned to receive Ipilimumab plus glycoprotein 100 (gp100;also known as melanocyte protein) (403 patients), Ipilimumab alone(137), or gp100 alone (136). The median overall survival was 10.0 monthsamong patients receiving Ipilimumab plus gp100, as compared with 6.4months among patients receiving gp100 alone (hazard ratio for death,0.68; P<0.001). The median overall survival with Ipilimumab alone was10.1 months (hazard ratio for death in the comparison with gp100 alone,0.66; P=0.003). No difference in overall survival was detected betweenthe Ipilimumab groups (hazard ratio with Ipilimumab plus gp100, 1.04;P=0.76) (see, for example, Hodi, F. S., et al. (2010) N Engl J Med 363,711-723). Following the success of anti-CTLA-4 therapy, antibodiestargeting programmed cell death protein 1 (PD-1), or its ligand PD-L1,proved to be effective at improving overall survival in a wide varietyof cancers (see, for example, Hamid, O., et al. (2013) N Engl J Med 369,134-144; Herbst, R. S., et al. (2014) Nature 515, 563-567; Powles, T.,et al. (2014) Nature 515, 558-562; Topalian, S. L., et al. (2014) J ClinOncol 32, 1020-1030; Ribas, A., and Wolchok, J. D. (2018) Science 359,1350-1355). For example, in one study, 296 patients received anti-PD-1ligand antibody treatment. Among 236 patients in whom a response couldbe evaluated, objective responses (complete or partial responses) wereobserved in those with non-small-cell lung cancer, melanoma, orrenal-cell cancer. Cumulative response rates (all doses) were 18% amongpatients with non-small-cell lung cancer (14 of 76 patients), 28% amongpatients with melanoma (26 of 94 patients), and 27% among patients withrenal-cell cancer (9 of 33 patients). Responses were durable; 20 of 31responses lasted 1 year or more in patients with 1 year or more offollow-up (see, Topalian, S. L., et al. (2012) N Engl J Med 366,2443-2454). The encouraging results of these studies has sparked aninterest from the cancer research field and inspired furtherinvestigations into targeting of alternative immune checkpointmolecules.

While checkpoint blockade represents a breakthrough in cancer therapy, amajority of cancer patients do not respond to these treatments, and sometumor types appear to be intrinsically resistant. The treatment isdesigned to boost an ongoing immune response and is inefficient in caseswhere initial immune activation is lacking, including tumors that aredevoid of infiltrating T-cells. Development of therapeutic strategies toenhance immune cell recruitment may therefore increase the proportion ofpatients responding to immune checkpoint blockade. Limitations ofcheckpoint inhibitors include systemic exposure of the patient to theantibodies used, as well as inability to consistently induce responses.

TNFα (Tumor Necrosis Factor-alpha or herein interchangeably referred toas TNF) promotes anti-cancer activity and as its name implies, is apotent cytokine initially characterized as an anti-tumor agent (see, forexample, Carswell, E. A., et al. (1975) Proc Natl Acad Sci USA 72,3666-3670). Subsequently, TNF was shown to have both pro-tumor andanti-tumor effects depending on its contextual activity within the tumormicroenvironment (see, for example, Wang, X., and Lin, Y. (2008) ActaPharmacol Sin 29, 1275-1288). In the tumor microenvironment, expressionof TNF at low levels contributes to angiogenesis, vessel permeability,and metastatic potential; whereas at high levels and during therapeuticdelivery to tumors, TNF has shown anti-tumor effects includingdisruption of vascular integrity through apoptosis, direct tumorkilling, and induction of anti-tumor immune responses (see, for example,Berberoglu, U., et al. (2004) Int J Biol Markers 19, 130-134; Michalaki,V., et al. (2004) Br J Cancer 90, 2312-2316; Talmadge, J. E., et al.(1987) Cancer Res 47, 2563-2570). Beneficial effects of elevated TNF inthe clinical setting have been reported. For example, a study of TNFexpression in 61 non-small cell lung carcinoma patients demonstratedexpression of TNF in 45.9% of cases that directly correlated with a morefavorable clinical outcome (see, for example, Boldrini, L., et al., G.(2000) Br J Cancer 83, 480-486). TNF administration is approved forisolated limb administration and has shown clinical benefit in isolatedhepatic procedures for liver cancer.

sTNF-Rs (soluble receptors of TNF) inhibit anti-cancer immune responsesand contribute to the control of TNF toxicity. The natural control orattenuation of TNF anti-tumor effects are attributed to the presence ofinhibitory molecules comprising shed soluble TNF receptors that arepresent in the plasma and bind/neutralize TNF (see, for example,Xanthoulea, S., et al. (2004) J Exp Med 200, 367-376; Aderka, D., et al.(1998) J Clin Invest 101, 650-659; Aderka, D., et al. (1991) Cancer Res51, 5602-5607; Selinsky, C. L., et al. (1998) Immunology 94, 88-93;Selinsky, C. L., and Howell, M. D. (2000) Cell Immunol 200, 81-87). Thecancer promoting activities of these soluble inhibitors was discoveredafter initial observations of cancer regressions that occurred inpatients undergoing plasmapheresis (see, for example, Israel, L., et al.(1976) Lancet 2, 642-643; Israel, L., et al. (1977) Cancer 40,3146-3154). Subsequent studies showed that this observation wasattributed to the removal sTNF-Rs. The molecular cloning of the cDNA andstudies of the recombinant proteins confirmed their anti-TNF activityand pro-tumor function (see, for example, Schall, T. J., et al. (1990)Cell 61, 361-370; Engelmann, H., et al. (1990) J Biol Chem 265,1531-1536).

At low doses of TNF, the normal concentrations of sTNF-R inhibitors canbind and inactivate the small amounts of administered TNF. However,dosing of increased amounts or the stimulation of higher TNF productionto higher than normal levels can induce sTNF-R shedding that counteractsthe ability of TNF to reach therapeutic anti-tumor concentrationswithout toxic effects. Thus, the ability to overcome TNF inhibition toachieve anti-cancer effects requires administration of TNF in amountsthat are much too close to the maximum tolerated dose (MTD). For thisreason, systemic TNF therapy, although possibly effective, has showntoxicity in numerous human clinical trials. Due to this adverserisk/benefit consideration, systemic therapy using TNF has largely beenabandoned. However, isolated limb procedures that block systemicexposure to TNF have been performed in combination with chemotherapeuticagents (see, for example, Deroose, J. P., et al. (2012) Ann Surg Oncol19, 627-635; Verhoef, C., et al. (2007) Curr Treat Options Oncol 8,417-427).

There have been attempts to use medical devices which extracorporeallyremove tumor produced immune “blocking factors.” Unfortunately, to date,these devices have suffered many limitations such as: a) non-specificbinding of other biological materials; b) “leaching” of immunoadsorptivematerials from the device into patients' circulation; and c) ineffectiveremoval of the target protein from circulation. The present inventionovercomes these and other limitations of prior systems.

SUMMARY

The following is a non-exhaustive listing of some aspects of the presenttechniques. These and other aspects are described in the followingdisclosure.

Accordingly, one or more aspects of the present disclosure relate to asystem for removing at least one target component of body fluid. Thesystem comprises: an inlet configured to receive the body fluid from apatient and a sequestering chamber coupled to the inlet and configuredto receive the body fluid from the inlet. The sequestering chambercomprises a capture support configured to bind to the at least onetarget component of the body fluid to capture the at least one targetcomponent in the sequestering chamber responsive to contact between thecapture support and the body fluid. The capture support is configured tobind to the at least one target component to reduce an amount of the atleast one target component in the body fluid. The sequestering chambercomprises first and second access ports configured to provide access tothe sequestering chamber separate from the inlet. The first and secondaccess ports are configured to facilitate insertion and/or removal ofthe capture support to and/or from the sequestering chamber. The systemcomprises an outlet configured to pass the body fluid having the reducedamount of the at least one target component from the sequesteringchamber for optional reintroduction of some or all of the body fluidhaving the reduced amount of the at least one target component back intothe patient; and one or more filters configured to separate the capturesupport in the sequestering chamber from the inlet and the outlet. Theone or more filters are configured to retain the capture support withinthe sequestering chamber.

In an embodiment, (a) a capture efficiency of the capture supportbinding to the at least one target component is 80% or more at a flowrate of 45 mL per minute of plasma flow or less, and optionally (b) abinding affinity of the capture support to the at least one targetcomponent is at least about 10⁻⁷ K_(D) and/or (c) a leach rate of thecapture support through the outlet is less than about 100 ng/mL/min.

In an embodiment, (b) a binding affinity of the capture support to theat least one target component is 10⁻⁷ K_(D) or greater, and optionally(a) a capture efficiency of the capture support binding to the at leastone target component is 80% or more at a flow rate of 45 mL/min or less,and/or (c) a leach rate of the capture support through the outlet isless than about 100 ng/mL/min.

In an embodiment, (c) a leach rate of the capture support through theoutlet is less than about 100 ng/mL/min, and optionally (a) a captureefficiency of the capture support binding to the at least one targetcomponent is 80% or more at a flow rate of 45 mL/min or less, and/or (b)a binding affinity of the capture support to the at least one targetcomponent is 10⁻⁷ K_(D) or greater.

In an embodiment, the body fluid comprises plasma.

In an embodiment, the at least one target component comprises a protein,complex, assembly, or cell.

In an embodiment, the at least one target component comprises one ormore plasma components that function to inhibit anti-cancer immuneresponses in the patient.

In an embodiment, the at least one target component comprises one ormore immune inhibitors.

In an embodiment, the at least one target component comprises a solubleTNF-α receptor.

In an embodiment, the at least one target component comprises an sTNF-R1receptor and/or an sTNF-R2 receptor.

In an embodiment, the capture support comprises an affinitychromatography support material. In other embodiments, the capturesupport comprises hollow fiber membranes, sheet or rolled sheetmembranes, membrane cassettes, and/or beads.

In an embodiment, the capture support comprises the affinitychromatography support material, and the affinity chromatography supportmaterial comprises sepharose, agarose, or acrylamide.

In an embodiment, the capture support comprises a porous or non-porousmatrix material including, but not limited to, ceramic material.

In an embodiment, the capture support is configured to bind to more thanone target component of the body fluid.

In an embodiment, the capture support comprises a solid support havingantibodies, antibody fragments, binding peptides, aptamers, or avimersimmobilized thereon.

In an embodiment, the antibodies are selected from the group consistingof IgA, IgD, IgE, IgG, IgM, and combinations thereof.

In an embodiment, the capture support comprises TNFα, multimers of TNFα,single chain TNFα, fragments of TNFα, multimers of fragments of TNFα, orcombinations thereof.

In an embodiment, multimers of TNFα comprise TNFα monomers in which oneor more monomers is in an amino terminal to carboxyl terminal linkage.

In certain embodiments, multimers of TNFα can exclude or include aspacer between the monomers.

In certain embodiments, a spacer comprises one or more amino acidresidues.

In an embodiment a spacer comprises one or more glycine, serine and/oralanine amino acids.

In an embodiment, the capture support comprises an sc-TNFα ligand,optionally the entire sequence or partial sequence of SEQ ID NO:1, SEQID NO:2, or SEQ: ID NO:3.

In an embodiment, the capture support comprises a trimeric form of theTNFα ligand.

In an embodiment, the trimeric form of the scTNFα ligand comprises thesequence of SEQ ID NO:2 or SEQ: ID NO:3, with or without the spaceramino acids.

In an embodiment, the capture support comprises ligands bound to beads.

In an embodiment, the ligands have a given density and orientation on agiven bead. The density and/or orientation is configured to enhancebinding between the ligands and the at least one target component of thebody fluid.

In an embodiment, a size, number, density, and/or concentration of thebeads is configured to facilitate a laminar flow of the body fluidthrough the beads to enhance the binding between the ligands and the atleast one target component of the body fluid.

In an embodiment, the beads are quenched in ethanolamine to enhancebinding specificity.

In an embodiment, the body fluid is whole blood.

In an embodiment, the inlet, the sequestering chamber, and the outletform an extracorporeal closed-circuit column.

In an embodiment, the extracorporeal closed-circuit column is configuredto remain sterile during operation.

In an embodiment, the system comprises a target component outlet portconfigured to facilitate sampling or removal of all or part of thecaptured at least one target component without compromising theextracorporeal closed-circuit column.

In an embodiment, the system comprises an elution reagent portconfigured to facilitate introduction of an elution reagent into thesequestering chamber without compromising the extracorporealclosed-circuit column.

In an embodiment, the elution reagent port is further configured toreceive a conditioning agent configured to prepare the system for reuse.

In an embodiment, the system further comprises a pump configured todrive a reconditioning agent through the inlet, the sequesteringchamber, and the outlet.

In an embodiment, the pump comprises a syringe pump, a peristaltic pump,a piston pump, a diaphragm pump, or a combination thereof.

In an embodiment, the one or more filters have an average pore diameterbetween about 3 microns and about 100 microns.

In an embodiment, the system further comprises one or more additionalsequestering chambers including capture supports having the samefunctionality.

In an embodiment, the one or more additional sequestering chamberscombine with the sequestering chamber to form a multistage separationcircuit configured to bind with a plurality of different targetcomponents.

In an embodiment, the patient is a human or veterinary subject. Theveterinary subject may include domestic animals such as dogs, cats,etc.; farm or ranch animals such as equine, porcine, bovine, etc.;and/or other animals.

According to another embodiment, a method for removing the at least onetarget component of the body fluid with the system of any of theembodiments described above is provided. The method comprises:conducting the body fluid from the patient through the inlet to thesequestering chamber; binding the at least one target component of thebody fluid to capture the at least one target component in thesequestering chamber to reduce the amount of the at least one targetcomponent in the body fluid; and optionally passing some or all of thebody fluid having the reduced amount of the at least one targetcomponent from the sequestering chamber through the outlet forreintroduction back into the patient.

In an embodiment, the method further comprises measuring the reducedamount of the at least one target component in the body fluidreintroduced back into the patient.

In an embodiment, the measuring comprises one or more of liquidchromatography—mass spectrometry (LC-MS), high performance liquidchromatography (HPLC), ultra-high performance liquid chromatography(UHPLC), resistance measurements, light emission measurements,chemiluminescence, electroluminescence, electrochemiluminescence,chromatographic monitoring, positron emission tomography (PET), x-raycomputed tomography (CT), magnetic resonance imaging (MRI), ultrasound,gamma camera, single photon emission computed tomography (SPECT), ELISA,surface plasmon resonance (SPR) and/or biolayer interferometry (BLI).

In an embodiment, the method further comprises measuring a leach rate ofthe capture support in the body fluid reintroduced back into thepatient.

In some embodiments, the method is for human, veterinary,domestic/companion animal, ranch/farm animal, and/or other use.

These and other objects, features, and characteristics of the system ormethod disclosed herein, as well as the methods of operation andfunctions of the related elements of structure and the combination ofparts and economies of manufacture, will become more apparent uponconsideration of the following description and the appended claims withreference to the accompanying drawings, all of which form a part of thisspecification, wherein like reference numerals designate correspondingparts in the various figures. It is to be expressly understood, however,that the drawings are for the purpose of illustration and descriptiononly and are not intended as a definition of the limits of theinvention. As used in the specification and in the claims, the singularform of “a”, “an”, and “the” include plural referents unless the contextclearly dictates otherwise.

BRIEF DESCRIPTION OF THE DRAWINGS

The above-mentioned aspects and other aspects of the present techniqueswill be better understood when the present application is read in viewof the following figures in which like numbers indicate similar oridentical elements.

FIG. 1 illustrates an example embodiment of the present system, inaccordance with one or more embodiments.

FIG. 2 provides a more detailed view of the present system, inaccordance with one or more embodiments.

FIG. 3 illustrates a corresponding cross-sectional view of the presentsystem, in accordance with one or more embodiments.

FIG. 4 illustrates a capture support (e.g., ligand coated beads in thisexample) in a sequestering chamber of a housing of the system binding toa target component of body fluid (plasma in this example) to capture thetarget component in the sequestering chamber, in accordance with one ormore embodiments.

FIG. 5 is an enlarged view of the capture support shown in FIG. 4, inaccordance with one or more embodiments. The enlarged view shows thecapture support comprising a bead and capture ligands.

FIG. 6 illustrates an example regeneration mechanism, in accordance withone or more embodiments.

FIG. 7 illustrates example embodiments where the system includes two ormore internal sequestering chambers, which can be configured in seriesor parallel relative to fluid flow from inlet to outlet, in accordancewith one or more embodiments.

FIG. 8 illustrates example methods of use where multiple systems can beutilized in series and/or parallel configurations within a singleplasmapheresis flow circuit, in accordance with one or more embodiments.

FIG. 9 illustrates multiple sequestering chambers, each withindependently controllable flow valves, in a single housing, inaccordance with one or more embodiments

FIG. 10 illustrates a method for removing the target component of theblood, plasma, and/or other body fluid with the system, in accordancewith one or more embodiments.

FIG. 11 illustrates representative system performance characteristicsincluding sTNF-R1 and sTNF-R2 reduction from a patient's blood pool andcolumn capture efficiency as a function of procedure time, in accordancewith one or more embodiments.

While the invention is susceptible to various modifications andalternative forms, specific embodiments thereof are shown by way ofexample in the drawings and will herein be described in detail. Thedrawings may not be to scale. It should be understood, however, that thedrawings and detailed description thereto are not intended to limit theinvention to the particular form disclosed, but to the contrary, theintention is to cover all modifications, equivalents, and alternativesfalling within the spirit and scope of the present invention as definedby the appended claims.

DETAILED DESCRIPTION OF EXEMPLARY EMBODIMENTS

To mitigate the problems described herein, the inventors had to bothinvent solutions and, in some cases just as importantly, recognizeproblems overlooked (or not yet foreseen) by others in the field.Indeed, the inventors wish to emphasize the difficulty of recognizingthose problems that are nascent and will become much more apparent inthe future should industry trends continue as the inventors expect.Further, because multiple problems are addressed, many of themsimultaneously, it should be understood that some embodiments areproblem-specific, and not all embodiments address every problem withtraditional systems described herein or provide every benefit describedherein. That said, improvements that solve various permutations of theseproblems are described below.

The present system and method are useful for immune modulation of cancerpatients and may provide comparatively useful immune modulation forother diseases, including but not limited to, auto-immune andinflammatory disorders. In some embodiments, extracorporeal removal ofimmune suppressive factors from blood of patients using immunoadsorptivemeans is provided. In some embodiments, the present system and methodprovide for the efficient removal of soluble tumor necrosis factorreceptors (sTNF-Rs) from cancer patients. TNF is an endogenous cytokinethat modulates tumor growth and suppression as part of the body'snatural immune response to cancer. However, in many cancers, theanti-tumor effects of TNF are blocked by the presence of circulatinginhibitory molecules known as soluble TNF receptors (sTNF-R1 andsTNF-R2; see, for example, Gatanaga, T., et al. (1990) Lymphokine Res 9,225-229; Gatanaga, T., et al. (1990) Proc Natl Acad Sci USA 87,8781-8784; Schall, T. J., et al. (1990) Cell 61, 361-370; Berberoglu, U.et al. (2004) Int J Biol Markers 19, 130-134). These receptors, whichblock the therapeutic anti-tumor effects of endogenous TNF, have beenshown to increase in cancers and correlate with disease stage (see, forexample, Aderka, D., et al. (1991) Cancer Res 51, 5602-5607). sTNF-Rsare also a prognostic indicator for breast, malignant melanoma,colorectal, and bone sarcomas, and correlate negatively with patientsurvival (see, for example, Langkopf, F., and Atzpodien, J. (1994)Lancet 344, 57-58; Viac, J., et al. (1996) Eur J Cancer 32A, 447-449).Selective removal of sTNF-Rs from the patient's blood via plasmaapheresis, a process known as Immunopheresis™ enhances the patient'snatural anti-tumor immune response by unmasking the anti-tumor effectsof endogenous TNF, which can facilitate reduction of tumor burden andimprove patient survival.

FIG. 1 illustrates an example embodiment of the present system, (item10). In FIG. 1, system 10 is shown coupled to an apheresis machine 12.System 10 is configured to selectively remove sTNF-Rs (e.g., solubleTumor Necrosis Factor Receptor 1 (sTNF-R1), also known as tumor necrosisfactor receptor superfamily member 1A (TNFRSF1A and CD120a); and solubleTumor Necrosis Factor Receptor 2 (sTNF-R2), also known as tumor necrosisfactor receptor superfamily member 1B (TNFRSF1B and CD120b), as exampletarget components, from the blood of a cancer patient 14. System 10includes a highly-selective binding matrix within a housing 16 that hasvarious ports that facilitate filling and plasma flow during use. Blooddrawn from patient 14 may be processed to obtain plasma, and the plasmatreated by placing system 10 into a plasma flow line 18 of apheresismachine 12 for a plasmapheresis procedure.

Examples of commercially available apheresis machines 12 include aTerumo BCT Spectra Optia System, for example. Other manufacturers ofapheresis machines include, but are not limited to, Fresenius,Haemonetics, Baxter, Nigale and Asahi. Apheresis may then be performedin accordance with the manufacturer's instructions.

As shown in FIG. 1, apheresis machine 12 may facilitate intravenousremoval of blood 20 from patient 14 and then separation 22 of the bloodinto plasma and cell fractions (e.g., using centrifugal forces, amembrane filter, and/or other components). The plasma fraction is thenpumped into system 10 where the plasma passes through a capture support(that includes a binding matrix as described herein) that captures, forexample, sTNF-Rs using a TNF ligand (as described herein). The plasma isthen pumped back out of system 10, where some or all of the treatedplasma may be recombined with the separated cells of patient 14, andthen reintroduced 24 back into the circulatory system of patient 14.

In some embodiments, treated plasma may be discarded and replaced byfresh plasma. For example, the plasma exchange may be concurrent wherethe exchange plasma is further treated to remove inhibitors.Immunopheresis (e.g., as described herein) could be done followingplasma exchange, for example.

It should be noted that although blood, plasma, sTNF-R's and TNF ligandsare specifically mentioned throughout this application, the componentsand/or principles described herein may be applied for other body fluids,other target components of a body fluid, and/or other capturing orbinding elements.

FIG. 2 provides a more detailed view of system 10. FIG. 3 illustrates acorresponding cross-sectional view. Referring to FIG. 2 and FIG. 3,system 10 comprises housing 16, an inlet 200, a sequestering chamber 202that includes a capture support 204, access ports 206, 208, an outlet210, one or more filters (e.g., 212 and 214 shown in FIG. 3), end caps250 and 252, and/or other components. As shown in FIG. 2 and FIG. 3,system 10 may form an extracorporeal closed-circuit column, for example.

The closed-circuit column may be configured to remain sterile duringoperation. In some embodiments, the components of the system 10 arewashed, for example, with 70% isopropyl alcohol prior to assembly toremove particulates. End caps 250 and 252 may be fitted with filters 212and 214, and pressed onto the ends of a barrel or tube (for example)forming housing 16. Caps 254 and 256 may be screwed into and/orotherwise coupled with inlet 200 and outlet 210. This sub-assembly maybe packaged and sterilized, for example, using ethylene oxide (EtO). EtOresiduals may be allowed to dissipate prior to continuing productionsteps. The EtO sterilized subassembly may be aseptically filled withcapture support 204 through access ports 206 and 208, and then ports 206and 208 may be securely capped with polycarbonate (for example) Luer(for example) caps (e.g., as described herein). An assembled device maythen be individually packaged and terminally sterilized using E-beamirradiation using 17.5-30 kGy (for example). In some embodiments, othermeans of sterilization that may be utilized include gamma irradiation,ethylene oxide, hydrogen peroxide, bleach, heat sterilization, steamsterilization, ozone and/or other sterilization operations depending onthe stability of capture support 204 and/or other factors.

Housing 16, inlet 200, outlet 210, and/or other components of system 10may be configured to couple with a (e.g., plasma) flow line of anapheresis machine (e.g., machine 12 as shown in FIG. 1). Housing 16,inlet 200, outlet 210, and/or other components of system 10 may beconfigured to couple with the flow line of the apheresis machine at apoint in the flow line that is after the patient's blood has beenseparated (e.g., as described herein) into cellular and plasmafractions, for example. Housing 16 may form a fluid channel or flow pathto conduct the body fluid of a patient between inlet 200 and outlet 210.

Housing 16 may provide structural support for capture support 204 and/orother components of system 10. Housing 16 may form an elongated tubularbody having a circular cross-sectional shape, and/or othercross-sectional shapes. Housing 16 may house sequestering chamber 202including capture support 204, one or more filters 212 and/or 214,and/or other components. In some embodiments, housing 16 and/or othercomponents of system 10 may be manufactured by injection molding and/orother operations.

Housing 16 may house filters 212 and 214 such that filters 212 and 214are substantially perpendicular to a fluid flow direction between inlet200 and outlet 210. Filters 212 and 214 may be configured to separatecapture support 204 in the sequestering chamber 202 from inlet 200 andoutlet 210. Filters 212, 214 may be configured to retain capture support204 within sequestering chamber 202, and/or perform other functions.

In some embodiments, filters 212 and/or 214 may form porous barriersmounted substantially perpendicular to a direction of fluid flow throughhousing 16. Filter 212 may be located proximate to inlet 200, and filter214 may be located proximate to outlet 210, thereby forming sequesteringchamber 202 inside housing 16. Filters 212 and/or 214 may be configuredto prevent portions (up to and including all) of capture support 204(e.g., one or more beads as described herein) from escaping system 10and passing into a patient's circulatory system, for example. In someembodiments, filters 212 and/or 214 may comprise porous frits, forexample. In some embodiments, filters 212, 214 may have an average porediameter between about 3 microns and about 100 microns, for example,and/or other average pore diameters. In some embodiments, filters 212and 214 may have a diameter that is larger than an inner diameter ofhousing 16 such that filters 212 and 214 fit snugly within housing 16and do not move when body fluid flows through system 10. In someembodiments, filters 212 and 214 are held in place by pressure from endcaps 250 and 252 (described below) pressing filters 212 and 214 againstrims of housing 16 (e.g., at either end of a tube formed by housing 16).In some embodiments, filters 212 and 214 are held in place withinhousing 16 by other mechanisms such as adhesives, washers, gaskets,stitching, over-molding, ultrasonic welding, and/or other componentsand/or processes. In some embodiments, filters 212 and 214 may be formedfrom polyethylene and/or other materials.

In some embodiments, housing 16 includes end caps 250 and 252 at eitherend of housing 16 that form and/or include inlet 200 and outlet 210. Endcaps 250 and 252 may be threaded to housing 16 and/or be coupled tohousing 16 in other ways (e.g., via clips, clamps, adhesive, ultrasonicwelding, pressure fitted, etc.). In some embodiments, end caps 250and/or 252 may be affixed onto housing 16 to ensure system 10 issubstantially airtight. In other words, system 10 is configured towithstand internal and external pressure forces (both air and fluid) toensure sterility during storage, shipping, and use. In some embodiments,end caps 250 and 252 terminate at inlet 200 and outlet 210,respectively. In some embodiments, inlet 200 and/or outlet 210 mayinclude caps 254, 256. In some embodiments, end caps 250 and/or 252 maybe formed from polypropylene and/or other materials. In someembodiments, caps 254 and/or 256 may be formed from high densitypolyethylene and/or other materials, for example.

In some embodiments, housing 16 may be formed from plastic and/or othermaterials. For example, housing 16 may be formed from one or more ofECTFE (ethylene-chlorotrifuluoroethylene copolymer, halar ECTFE, ETFE(ethylene-tetrafluoroethylene), tefzel ethylene tetrafluoroethylene(ETFE), FEP (fluorinated ethylene polypropylene), HDPE (high densitypolyethylene), LDPE (low density polyethylene), PC (polycarbonate),Makrolon polycarbonate, PEI (polytheterimide), PET (polyethyleneterephthalate), PETG (polyethylene terephthalate copolymer), PFA(polyfluoroalkoxy), Teflon PFA, PMMA (polymethyl methacrylate), PMP(polymethypentene), polypropylene, PPCO (polypropylene copolymer),polystyrene, PSF (polysulfone), PTFE (polytetrafluoroethylene), SAN(styrene acrylonitrile), TFE (tetrafluoroethlene), Teflon TFE, TMX(thermanox), PMX (permanox), and/or other materials. In someembodiments, housing 16 may be formed from metallic materials (e.g.,iron, iron alloy, steel, stainless steel, aluminum, aluminum alloy),glass and/or other materials.

Inlet 200 may be configured to receive blood, plasma, and/or other bodyfluids from a patient (e.g., patient 14 shown in FIG. 1). Outlet 210 maybe configured to pass blood, plasma, and/or other body fluid having areduced amount of one or more target components from sequesteringchamber 202 for reintroduction back into the patient (e.g., patient 14shown in FIG. 1). In some embodiments, inlet 200 and/or outlet 210 maybe a Luer fitting and/or other inlet or outlet fluidic connector typesor configurations. In some embodiments, inlet 200 and/or outlet 210 areconfigured to be fluidically coupled to apheresis machine tubing sets,intravenous tubing extension sets, fluidic tubing adapters, filters,stopcocks, and/or other elements commonly used in closed-loop patientfluid line assemblies. In some embodiments, inlet 200 and/or outlet 210may be configured such that the blood, plasma, and/or other body fluidsfrom a patient flow through system 10 at a flow rate of between about 5mL/min and about 300 mL/min, and/or other flow rates. In someembodiments, the flow rate may be between about 10 mL/min and about 100mL/min. In some embodiments, the flow rate may be between about 25mL/min and about 75 mL/min. In some embodiments, the flow rate may bebetween about 35 mL/min and about 70 mL/min. In some embodiments, theflow rate may be between about 40 mL/min and about 60 mL/min. Theseexemplary flow rates are in the range that can be accommodated by thesystem described herein. For some procedures, flow rates of less than 5mL/min may require an inordinate amount of time to complete. Conversely,flow rates of over 300 mL/min may limit capture efficiency of the system10 when used in conjunction with an apheresis machine. However, it isanticipated that flow rates of 300 mL/min are possible. Inlet 200 and/oroutlet 210 may have a diameter of a specific size and/or other featuresthat facilitate such flow rates.

In some embodiments, inlet 200 and/or outlet 210 may be configured suchthat human plasma containing up to about 200 micrograms (for example) ofsTNF-R proteins flow through system 10. This example is based on anexpected total plasma amount of sTNF-Rs of the patient. Theconcentration range of sTNF-Rs (combined sTNF-R1 and sTNF-R2) in humanplasma is approximately 3-10 ng/mL and the plasma volume of an examplepatient may be in the range of 50 cc per Kg body weight (W). The totalamount of sTNF-R is about (W(Kg)×50 mL/Kg×(3-10 ng/mL)/1000 ng/μg).Thus, for an individual of 70 Kg (for example), the amount of sTNF-Rswould be in the range of 10.5 to 35 micrograms. In some embodiments, anexcess amount of capture capability of system 10 may create an amplemargin of efficiency based on laboratory bench testing of excessiveamounts of sTNF-Rs with plasma flow through system 10 at rates of up toabout 45 mL/min (for example). In some embodiments, inlet 200 and/oroutlet 210 may be formed from polypropylene, polycarbonate, Makrolon™and/or other materials.

Sequestering chamber 202 may be coupled to inlet 200. Sequesteringchamber 202 may be configured to receive the blood (e.g., whole blood),plasma, and/or other body fluid from inlet 200. Sequestering chamber 202may comprise capture support 204, access ports 206, 208, and/or othercomponents.

Capture support 204 may be configured to bind to at least one targetcomponent of the blood, plasma, and/or other body fluid to capture theat least one target component in sequestering chamber 202. Capturesupport 204 may be and/or include a binding matrix (comprising beadsligands and/or other components as described herein), for example. Thecapturing may occur responsive to contact between capture support 204and the blood, plasma, and/or other body fluid. Capture support 204 maybe configured to bind to the at least one target component to reduce anamount of the at least one target component in the blood, plasma, and/orother body fluid.

In some embodiments, the at least one target component may comprise acomplex, an assembly, or a cell. In some embodiments, the at least onetarget component may comprise one or more blood products such as plasmaor serum components that function to inhibit anti-cancer immuneresponses in the patient. In some embodiments, the at least one targetcomponent may comprise one or more immune inhibitors. For example, theat least one target component may comprise a soluble TNFα receptor, ansTNF-R1 receptor, an sTNF-R2 receptor, an sTNF-R1 and sTNF-R2 receptor,and/or other receptors and receptor combinations.

In some embodiments, the capture moiety may be selected so as to bind toand capture other specific molecules in the plasma. Examples of theseother molecules or targets are, but are not limited, to acetyl-cholinereceptors, adenosine receptors, adrenoreceptors, GABA receptors,angiotensin receptors, cannabinoid receptors, cholecystokinin receptors,dopamine receptors, glucagon receptors, glucocorticoid receptors,glutamate receptors, histamine receptors, mineralocorticoid receptors,olfactory receptors, opioid receptors, purinergic receptors, secretinreceptors, serotonin receptors, somatostatin receptors, steroid hormonereceptors, calcium-sensing receptors, hormone receptors, erythropoietinreceptors, and natriuretic peptide receptors or their ligands. Otherexamples include but are not limited to type I cytokine receptors suchas type I interleukin receptors, erythropoietin receptor, GM-CSFreceptor, G-CSF receptor growth hormone receptor, oncostatin M receptor,myostatin receptor, leukemia inhibitory factor receptor; type IIcytokine receptors such as type II interleukin receptors, interferon-α/βreceptors, interferon-γ receptor or their ligands; members of theimmunoglobulin superfamily such as interleukin-1 receptor, CSF1, ckitreceptor, interleukin-18 receptor or their ligands; CD27, CD40 andlymphotoxin receptor or their ligands; chemokine receptors includingserpentine CCR and CXCR receptors such as CCR1 and CXCR4, andinterleukin 8 receptor or their ligands; TGF β receptors including TGF βreceptor 1 and TGF β receptor 2 or their ligands; galectins; and/orother structures (see Ozaki and Leonard, J. Biol. Chem 277:29355-29353,2002).

In some embodiments, capture support 204 may comprise a solid supportand/or other components. The solid support may be an affinitychromatography support material, hollow fiber membranes, sheetmembranes, membrane cassettes, rolled sheet membranes, and/or othermaterials. In embodiments where capture support 204 comprises theaffinity chromatography support material, the affinity chromatographysupport material may comprise a sugar, carbohydrate or polysaccaharidesuch as sepharose, agarose, or a polymer such as acrylamide, and/orother materials.

In some embodiments, capture support 204 may comprise a porous ornon-porous matrix material. In some embodiments, capture support 204 maybe configured to bind to more than one target component of the blood,plasma, and/or other body fluid.

In some embodiments, capture support 204 may be an affinitychromatography matrix comprising different capture moieties includingbut not limited to affinity reagents (e.g., a ligand as describedherein) bound to a support. The affinity chromatography matrix mayalternatively comprise a linking group, such as, but not limited to,cyanogen bromide, tresyl, triazine, vinyl sulfone, an aldehyde, anepoxide, or an activated carboxylic acid to facilitate coupling of anaffinity reagent (e.g., a ligand) to the solid support. Thechromatography matrix may be prepared by coupling the methyl-lysineaffinity reagent to the solid support with a linking group by chemicallyactivating the solid support, if necessary, and contacting the solidsupport with the methyl-lysine affinity reagent such that the affinityreagent covalently attaches to the solid support. Additionally, theaffinity reagent may be coupled to the solid support through a linker tomake the affinity reagent more accessible for binding to methylatedproteins and peptides.

In some embodiments, capture support 204 may comprise a solid supporthaving antibodies, antibody fragments, binding peptides, aptamers,avimers, and/or other components immobilized thereon. In someembodiments, the antibodies are one or more of IgA, IgD, IgE, IgG, orIgM, immunoglobulin subclasses and mixtures thereof, combinationsthereof, and/or other antibodies.

In some embodiments, capture support 204 may comprise an affinityreagent comprising ligands such as TNFα (as described above, TNF andTNFα are used interchangeably herein), multimers of TNF, single chain(sc) TNF, fragments of TNF, multimers of fragments of TNF, orcombinations thereof. In some embodiments, capture support 204 may beand/or include TNF ligands bound to one or more solid supports. In someembodiments, the binding may be covalent linking and/or other binding,for example.

Types of TNF include mammalian TNF, such as primate TNF and human

TNF. Exemplary human TNF sequences comprise:

1. (SSSRTPSDKPVAHVVANPQAEGQLQWLNRRANALLANGVELRDNQLVVPSEGLYLIYSQVLFKGQGCPSTHVLLTHTISRIAVSYQTKVNLLSAIKSPCQRETPEGAEAKPWYEPIYLGGVFQLEKGDRLSAEINRPDYLDFAESGQVYFGIIAL, SEQ ID NO: 1) -[processed TNF monomer, from Genbank Accession No. AQY77150.1]; 2.Trimeric form: (MCGSHHHHHHGSASSSSRTPSDKPVAHVVANPQAEGQLQWLNRRANALLANGVELRDNQLVVPSEGLYLIYSQVLFKGQGCPSTHVLLTHTISRIAVSYQTKVNLLSAIKSPCQRETPEGAEAKPWYEPIYLGGVFQLEKGDRLSAEINRPDYLDFAESGQVYFGIIALGGGSGGGSGGGSGGGSSSRTPSDKPVAHVVANPQAEGQLQWLNRRANALLANGVELRDNQLVVPSEGLYLIYSQVLFKGQGCPSTHVLLTHTISRIAVSYQTKVNLLSAIKSPCQRETPEGAEAKPWYEPIYLGGVFQLEKGDRLSAEINRPDYLDFAESGQVYFGIIALGGGSGGGSGGGSGGGSSSRTPSDKPVAHVVANPQAEGQLQWLNRRANALLANGVELRDNQLVVPSEGLYLIYSQVLFKGQGCPSTHVLLTHTISRIAVSYQTKVNLLSAIKSPCQRETPEGAEAKPWYEPIYLGGVFQLEKGDRLSAEINRPDYLDFAESGQVYFGIIAL, SEQ ID NO: 2); and 3. Trimeric form:(GSASSSSRTPSDKPVAHVVANPQAEGQLQWLNR RANALLANGVELRDNQLVVPSEGLYLIYSQVLFKGQGCPSTHVLLTHTISRIAVSYQTKVNLLSAIKSPCQ RETPEGAEAKPWYEPIYLGGVFQLEKGDRLSAEINRPDYLDFAESGQVYFGIIALGGGSGGGSGGGSGG GSSSRTPSDKPVAHVVANPQAEGQLQWLNRRANALLANGVELRDNQLVVPSEGLYLIYSQVLFKGQGCP STHVLLTHTISRIAVSYQTKVNLLSAIKSPCQRETPEGAEAKPWYEPIYLGGVFQLEKGDRLSAEINRPDY LDFAESGQVYFGIIALGGGSGGGSGGGSGGGSSSRTPSDKPVAHVVANPQAEGQLQWLNRRANALLAN GVELRDNQLVVPSEGLYLIYSQVLFKGQGCPSTHVLLTHTISRIAVSYQTKVNLLSAIKSPCQRETPEGAE AKPWYEPIYLGGVFQLEKGDRLSAEINRPDYLDFAESGQVYFGIIAL, SEQ ID NO: 3).

Exemplary TNF can comprise a monomer of the sequence of SEQ ID NO:1, adimer of the sequence of SEQ ID NO:1, a trimer of the sequence of SEQ IDNO:1 or the timeric form, SEQ ID NO:2 or SEQ ID NO:3, or a partialsequence thereof. The monomers comprising SEQ ID NO:2 or SEQ ID NO:3 mayoptionally be covalently linked by a spacer sequence of glycines orserines, such as GGGS, or spacer multimers such as (GGGS)₄. Amino acids,spacer sequences and spacer multimers may or may not be incorporatedinto dimeric or trimeric forms.

Naturally and non-naturally occurring variants of TNF are included. Suchvariants include gain and loss of function variants.

Non-limiting examples of TNF variants include one or more amino acidsubstitutions (e.g., 1-3, 3-5, 5-10, 10-15, 15-20, 20-25, 25-30, 30-40,40-50, 50-100, or more residues), additions (e.g., insertions or 1-3,3-5, 5-10, 10-15, 15-20, 20-25, 25-30, 30-40, 40-50, 50-100, or moreresidues) and deletions (e.g., subsequences or fragments) of a referenceTNF sequence. In some embodiments, a variant TNF sequence retains atleast part of a function or an activity of unmodified sequence, such asthe ability to bind to sTNF-R's (e.g., sTNF-R1 receptor and/or sTNF-R2receptor).

A variant can have one or more non-conservative or a conservative aminoacid sequence differences or modifications, or both. A “conservativesubstitution” is the replacement of one amino acid by a biologically,chemically or structurally similar residue. Biologically similar meansthat the substitution does not destroy a biological activity.Structurally similar means that the amino acids have side chains withsimilar length, such as alanine, glycine and serine, or a similar size.Chemical similarity means that the residues have the same charge or areboth hydrophilic or hydrophobic. Particular examples include thesubstitution of one hydrophobic residue, such as isoleucine, valine,leucine or methionine for another, or the substitution of one polarresidue for another, such as the substitution of arginine for lysine,glutamic for aspartic acids, or glutamine for asparagine, serine forthreonine, and the like. Particular examples of conservativesubstitutions include the substitution of a hydrophobic residue such asisoleucine, valine, leucine or methionine for another, the substitutionof a polar residue for another, such as the substitution of arginine forlysine, glutamic for aspartic acids, or glutamine for asparagine, andthe like. For example, conservative amino acid substitutions typicallyinclude substitutions within the following groups: glycine, alanine;valine, isoleucine, leucine; aspartic acid, glutamic acid; asparagine,glutamine; serine, threonine; lysine, arginine; and phenylalanine,tyrosine. A “conservative substitution” also includes the use of asubstituted amino acid in place of an unsubstituted parent amino acid.

Such variants include proteins or polypeptides which have been or may bemodified using recombinant DNA technology such that the protein orpolypeptide possesses altered or additional properties, for example.Variants can differ from a reference sequence, such as naturallyoccurring proteins or peptides.

At the amino acid sequence level, a naturally or non-naturally occurringvariant protein will typically be at least about 70% identical, moretypically about 80% identical, even more typically about 90% or moreidentity to the reference protein, although substantial regions ofnon-identity are permitted in non-conserved regions (e.g., less, than70% identical, such as less than 60%, 50% or even 40%). In otherembodiments, the sequences have at least 60%, 70%, 75% or more identity(e.g., 80%, 85% 90%, 95%, 96%, 97%, 98%, 99% or more identity) to areference sequence. Procedures for the introduction of amino acidchanges in a protein or polypeptide are known to the skilled artisan(see, e.g., Sambrook et al., Molecular Cloning: A Laboratory Manual(2007)).

The term “identity” and grammatical variations thereof, mean that two ormore referenced entities are the same, when they are “aligned”sequences. Thus, by way of example, when two polypeptide sequences areidentical, they have the same amino acid sequence, at least within thereferenced region or portion. The identity can be over a defined area(region or domain) of the sequence. An “area” or “region” of identityrefers to a portion of two or more referenced entities that are thesame. Thus, where two protein sequences are identical over one or moresequence areas or regions they share identity within that region. An“aligned” sequence refers to multiple protein (amino acid) sequences,often containing corrections for missing amino acids (gaps) as comparedto a reference sequence.

The identity can extend over the entire sequence length or a portion ofthe sequence. For example, the length of the sequence sharing thepercent identity is 2, 3, 4, 5 or more contiguous amino acids or more,e.g., 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, etc.contiguous amino acids. In another nonlimiting example, the length ofthe sequence sharing identity is 20 or more contiguous amino acids ormore, e.g., 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35,etc. contiguous amino acids. In a further nonlimiting example, thelength of the sequence sharing identity is 35 or more contiguous aminoacids, e.g., 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 45, 47, 48, 49, 50,etc., contiguous amino acids. In yet further particular nonlimitingexamples, the length of the sequence sharing identity is 50 or moreamino acids, e.g., 50-55, 55-60, 60-65, 65-70, 70-75, 75-80, 80-85,85-90, 90-95, 95-100, 100-110, etc. contiguous amino acids.

The extent of identity between two sequences can be ascertained using acomputer program and mathematical algorithm. Such algorithms thatcalculate percent sequence identity generally account for sequence gapsand mismatches over the comparison region or area. For example, a BLAST(e.g., BLAST 2.0) search algorithm (see, e.g., Altschul et al., J. Mol.Biol. 215:403 (1990), publicly available through NCBI) has exemplarysearch parameters as follows: Mismatch-2; gap open 5; gap extension 2.For protein or polypeptide sequence comparisons, a BLASTP algorithm istypically used in combination with a scoring matrix, such as PAM100, PAM250, BLOSUM 62 or BLOSUM 50. FASTA (e.g., FASTA2 and FASTA3) and SSEARCHsequence comparison programs are also used to quantitate extent ofidentity (Pearson et al., Proc. Natl. Acad. Sci. USA 85:2444 (1988);Pearson, Methods Mol Biol. 132:185 (2000); and Smith et al., J. Mol.Biol. 147:195 (1981)). Programs for quantitating protein structuralsimilarity using Delaunay-based topological mapping have also beendeveloped (Bostick et al., Biochem Biophys Res Commun. 304:320 (2003)).

Ligands and proteins, such as TNF, include additions and insertions, forexample, heterologous domains. An addition (e.g., heterologous domain)can be a covalent or non-covalent attachment of any type of molecule.Typically, additions and insertions (e.g., a heterologous domain) confera complementary or a distinct function or activity.

A nonlimiting example of an addition or insertion is an amino acidspacer, a spacer comprising two or more amino acids and multimers ofsuch spacers comprising two or more amino acids. Nonlimiting examples ofamino acid acids that function as amino acids and multimers of spacersinclude glycine, serine and alanine.

Additions and insertions include chimeric and fusion sequences, which isa protein sequence having one or more molecules not normally present ina reference native (wild type) sequence covalently attached to thesequence. The terms “fusion” or “chimeric” and grammatical variationsthereof, means that a portion or part of the molecule contains adifferent entity distinct (heterologous) from the molecule as they donot typically exist together in nature. That is, for example, oneportion of the fusion or chimera, includes or consists of a portion thatdoes not exist together in nature, and is structurally distinct.

In some embodiments, a method for covalently linking TNF ligands to thesolid support(s) comprises amine reductive chemistries, cyanogen bromide(CNBr), N-hydroxy succinimide esters, carbonyl diimidazole, reductiveamination, 2-fluoro-1-methylpyridinium (FMP) activation,1-ethyl-3-(3-dimethyiaminopropyl)carbodiimide (EDC)-mediated amide bondformation organic sulfonyl chlorides tosyl chloride and tresyl chloride,divinylsulfone, azlactone, cyanuric chloride (trichloror-s-triazine),sulfhydryl reactive chemistries, iodoacetyl and bromoacetyl activation,maleimide, pyridyl disulfide, divinysulfone, epoxy or bisoxiran,TNF-thiol, carbonyl reactive chemistries, hydrazide, reductiveamination, hydroxyl reactive chemistries, cyanuric chloride, activehydrogen reactive chemistries, diazonium, mannich condensation,photoreactive cross linking, immobilized serum albumin with CNBractivation, periodate activation, and/or other methods.

In some embodiments, the binding may be ionic binding, electrostaticbinding, Van der Waals binding, hydrophobic binding and/or otherbinding, for example. In some embodiments, an electrostatic bond may beformed between TNF ligands and the one or more solid supports usingliking molecules such as immobilized avidin streptavidin and monomericavidin bound to biotin, antibody-antigen complexes, ligand receptorcomplexes, and/or other linking molecules.

In some embodiments, the solid support may be formed from materials suchas agarose, sepharose, cellulose, pore glass, silica, acrylamidederivatives polyacrylamide beads, trisacryl, sephacryl, an Ultrogel® AcAchromatography sorbent (Pall Corporation), azlactone beads, methacrylatederivatives, a TSKgel® chromatography gel (Tosoh Corporation), aTOYOPEARL® HW polymer gel (Tosoh Corporration), HEMA (2 hydroxyethylmethacrylate, poly (2 hydroxyethyl methacrylate), Eupergit, polystyreneand its derivatives, Poros, polyether sulfone, a polysaccharide,polytetrafluoroethylene, polysulfone, polyester, polyvinylidenefluoride, polypropylene, poly (tetrafluoroethylene-co-perfluoro(alkylvinyl ether)), polycarbonate, polyethylene, glass, polyacrylate,polyacrylamide, poly(azolactone), polystyrene, polylactide, ceramic,nylon, metal, and/or other materials. In some embodiments, the solidsupport may be formed by plates, membranes, beads, ceramics, and/orother components.

In some embodiments, the solid support may be or include beads, forexample. In some embodiments, capture support 204 comprises ligandsbound to beads. As described above, in some embodiments, the ligands maybe and/or include sc (single chain)-TNFα ligands. In some embodiments,the ligands may be and/or include a dimeric or trimeric form of thesc-TNF ligand. In some embodiments, the ligands may be and/or include aTNF ligand such as a single-chain polypeptide (sc-TNF) ligand (monomer,dimer or trimer) that binds to, and effectively captures sTNF-Rs fromthe patient's plasma, for example.

Ligands that have conformational changes due to altered amino acidsequences or purity of the protein might be responsible for substantialchanges such as enhancing or reducing binding affinity. Such mutationswithin the sequence could respectively improve or decrease the bindingefficiency of the polypeptide to sTNF-Rs Impurities in TNF preparationswould lower the amount of TNF used for coupling by lowering the amountof TNF coupled in proportion to the total amount of protein that isused. These ligands may have high target affinity or binding affinityfor this target portion of the patient's biological material. Suchbinding affinities are represented as K_(D). (It should be noted thatthe lower the K_(D), value, the greater the binding affinity will be.)Representative target affinities of a ligand can be about, for example,greater than about 10⁻⁶ K_(D), or greater than about 10⁻⁷ K_(D), orgreater than about 10⁻⁸ K_(D), or greater than about 10⁻⁹ K_(D), orgreater than about 10⁻¹⁰ K_(D), or greater than about 10⁻¹¹ K_(D), orgreater than about 10⁻¹² K_(D), or greater than about 10⁻¹³ K_(D). Theaffinity of TNF for sTNF-R1 may be approximately 10⁻¹¹ K_(D), forexample. The affinity of TNF for sTNF-R2 may be approximately 10⁻¹⁰K_(D), for example.

In some embodiments, the sc-TNF ligands comprise sc-TNFα molecules. Insome embodiments, the sc-TNF ligands comprise a TNFα monomer, one ormore complexes of TNFα proteins, and/or other components. In someembodiments, the complexes comprise dimers, trimers, multimers, muteins,and/or fragments thereof. In some embodiments, capture support 204 maycomprise sc-TNF protein ligands conjugated to a plurality of agarosebeads (e.g., which selectively bind to sTNF-Rs present in the plasmathat is circulated through system 10, e.g. as shown in FIG. 1-3).

In some embodiments, generation of sc-TNF (and/or other ligands) may beperformed through various means of genetic engineering and proteinexpression (see, for example, Muller, R., et al. (1986) FEBS Lett 197,99-104; Mori, T., et al. (1994) Gene 144, 289-293; Horwitz, A. H., etal. (1996) Protein Expr Purif 8, 28-40; Li, H., et al. (2019) World JMicrobiol Biotechnol 35, 27; Ashman, K., et al. (1989) Protein Eng 2,387-391; Su, X., et al. (1992) Biotechniques 13, 756-762; Li, C. B., etal. (1992) Sci China B 35, 319-328; Guo, D., et al. (1995) BiochemBiophys Res Commun 207, 927-932; Xiang, J., et al. (1997) J Biotechnol53, 3-12 Tang, P., et al. (1996) Biochemistry 35, 8216-8225).

The ligands may have a given density and orientation on a given support(such as a bead, for example). The ligands can be covalently linked tothe beads (and/or other supports) through amines or thiol moieties, forexample. The density and orientation may be configured to enhancebinding between the ligands and the target component of the body fluid.In some embodiments, the ligands may be configured such that they extendout from the support matrix (e.g., a given bead) at the amino (N) orcarboxyl (C) terminal for binding accessibility. In some embodiments, alinker may be placed between the bead (for example) surface and theligand to extend the ligand into the body fluid passing through as ameans of reducing steric hindrance that would interfere with thebinding.

The density can be expressed in milligrams ligand per milligrams ofsupport. In some embodiments, the ligand may be a 54-K_(D) protein. Byway of example and without limitation, the support may have a liganddensity of at least about 0.1 mg (1.8×10⁻⁶ mmoles) ligand/mg of support(e.g., beads), at least about 1 mg (18×10⁻⁶ mmoles) ligand/mg of support(e.g., beads), at least about 5 mg ligand/mg of support (e.g., beads),at least about 7 mg (1.3×10⁻⁴ mmoles) ligand/mg of support (e.g.,beads), at least about 10 mg ligand/mg of support (e.g., beads), atleast about 15 mg ligand/mg of support (e.g., beads), or at least about20 mg ligand/mg of support (e.g., beads), or more, for example.

A size, number, density, and/or concentration of a support, such as abead or beads (e.g., in combination with a shape and size of housing16), may be configured to facilitate a laminar flow of the blood,plasma, and/or other body fluid through the beads to enhance the bindingbetween the ligands and the target component of the body fluid.Increasing a bead size may proportionally accommodate higher flow rates.Increasing the density of the coupled ligand may increase the capturecapacity while avoiding concentrations that may contribute sterichindrance which would interfere with effective binding of the targetmolecule. The size, number, density, orientation and/or concentration ofthe support (e.g., beads) may facilitate a flow-rate of plasma, forexample, through system 10 that effectively balances tradeoffs betweencapture rates and clinically practical procedure times. For example, aplasmapheresis procedure involving one embodiment of system 10 mayrequire circulation of two patient plasma volumes through thesequestering chamber 202 to achieve a specific target concentrationreduction of sTNF-R1/R2 from the patient's plasma; whereas analternative embodiment with twice the capture rate efficiency as thefirst embodiment may only require the circulation of one patient plasmavolume through its sequestering chamber to achieve comparable sTNF-R1/R2concentration reduction, thereby reducing the clinical procedure time byabout a factor of two. In some embodiments, the beads may include one ormore different bead materials such as commercially available agarose orpolyacrylamide compositions.

In some embodiments, the beads and/or other solid support may have asize that prevents them from passing through filters 212 and 214 (seefilter pore size discussion herein). In some embodiments, the beads maybe quenched (where the binding sites that are left after TNF couplingare saturated in their occupancy) with ethanolamine or ethylene diamineto enhance binding specificity. Ethanolamine may be used as a quenchingagent, for example, due to its biocompatibility profile. In someembodiments, the beads may be pretreated with agents such as immulons,polystyrenes or polyethylenes, in order to better control and maximizerecovery (or for other reasons), and/or the beads may be pre-treatedwith a commercially available cross-linker. A cross-linker may be anychemical or substance used to facilitate the attachment to the solidphase of the molecule that captures one or more circulating immunecomplexes. Non-limiting examples of commercially available cross-linkersare poly-L-lysine, glutaraldehyde, and cyanogen bromide, for example.

In some embodiments, the beads may form a binding matrix, which mayinclude covalently or non-covalently bound affinity molecules (e.g., asdescribed herein). In some embodiments, the beads may be in the range ofabout 20-1,000 μm in diameter, for example. In some embodiments, thebeads may be in the range of about 25-500 μm in diameter. In someembodiments, the beads may be in the range of about 25-200 μm indiameter. In some embodiments, the beads may be in the range of about40-180 μm in diameter. In some embodiments, the beads may be in therange of about 50-170 μm in diameter. In some embodiments, the beads maybe in the range of about 65-160 μm in diameter. In some embodiments, thebeads may be in the range of about 75-150 μm in diameter.

The beads may be formed from materials which are biocompatible and towhich various ligands are covalently linked or electrostatically bound(e.g., as described above). Binding of ligands may be performed usingcovalent binding methods such as amine reductive chemistries, cyanogenbromide (CNBr), N-hydroxy succinimide esters, carbonyl diimidazole,reductive amination, FMP activation, EDC-mediated amide bond formation,organic sulfonyl chlorides tosyl chloride and tresyl chloride,divinylsulfone, azlactone, cyanuric chloride (trichloro-s-triazine),sulfhydryl reactive chemistries, iodoacetyl and bromoacetyl activationmethods, maleimide, pyridyl disulfide, divinylsulfone, epoxy orbisoxiran, TNF-Thiol, carbonyl reactive chemistries, hydrazide,reductive amination, hydroxyl reactive chemistries, cyanuric chloride,active hydrogen reactive chemistries, diazonium, Mannich condensation,photoreactive cross linking, and/or other operations. Coupling can alsobe done using immobilized serum albumin with CNBr activation orperiodate activation. In some embodiments, binding may be performedusing non-covalent interactions such as a) immobilized avidinstreptavidin and monomeric avidin bound to biotin; b) antibody-antigencomplexes; and/or c) ligand-receptor complexes.

In some embodiments, a capture efficiency, equivalent to [1−(sTNF-Rplasma concentration at outlet 210/sTNF-R plasma concentration at inlet200)]×100 and thus expressed as a percentage, of capture support 204binding to the target component may be at least 10% or more sTNF-R1and/or sTNF-R2. In some embodiments, the capture efficiency may be atleast 50% or more. In some embodiments, the capture efficiency may be atleast 80% or more. In some embodiments, the capture efficiency may be atleast 90%, 95%, 96%, 97%, 98%, 99%, or more.

Capture efficiency values may take into consideration a time-basedcomponent (e.g., a capture efficiency over the first 5, 10, 15, 30, 45,60, 90, 120 or more minutes of a treatment) since available bindingsites within the system's capture matrix decrease as more target agentsare cumulatively captured within the column (system 10). By way ofseveral non-limiting examples, in some embodiments, at least 80% or moreof sTNF-R proteins (sTNF-R1 and/or sTNF-R2) in a flowing biologicalsample may become bound to TNF ligands within sequestering chamber 202within about 30 minutes, for example. In some embodiments, at least 90%or more of sTNF-R proteins (sTNF-R1 and/or sTNF-R2) in a flowingbiological sample may become bound to TNF ligands within sequesteringchamber 202 within about 30 minutes. In some embodiments, at least 95%or more of sTNF-R proteins (sTNF-R1 and/or sTNF-R2) in a flowingbiological sample may become bound to TNF ligands within sequesteringchamber 202 within about 30 minutes. In some embodiments, at least 96%or more of sTNF-R proteins (sTNF-R1 and/or sTNF-R2) in a flowingbiological sample may become bound to TNF ligands within sequesteringchamber 202 within about 30 minutes. In some embodiments, at least 97%or more of sTNF-R proteins (sTNF-R1 and/or sTNF-R2) in a flowingbiological sample may become bound to TNF ligands within sequesteringchamber 202 within about 30 minutes. In some embodiments, at least 98%or more of sTNF-R proteins (sTNF-R1 and/or sTNF-R2) in a flowingbiological sample may become bound to TNF ligands within sequesteringchamber 202 within about 30 minutes. In some embodiments, at least 99%or more of sTNF-R proteins (sTNF-R1 and/or sTNF-R2) in a flowingbiological sample may become bound to TNF ligands within sequesteringchamber 202 within about 30 minutes.

Although not wanting to be bound by any theory, the capture efficiencyvalues described herein may result from the use of the sc-TNF liganddescribed above (e.g., which has exceptionally high target affinity),the use of the trimeric form of the sc-TNF ligand, the purity of theligand, the ligand density on the beads, the ligand binding orientationon the beads, the size of the beads, the number, density andconcentration of the beads in system 10, a flow-rate through system 10that balances capture efficiency versus clinical procedure time, thephysical size and structure of housing 16 which yields the laminar flowthrough the beads, the (e.g., chemistry and/or electrostatic) processused for coupling the ligands to the beads, the sterilization techniqueand radiation dosage, and/or other factors. Put another way (again notwanting to be bound by any theory), one reason, for example, theefficiency of the column (system 10) is high may be due to the largeamount of capture ligand on the bead matrix in conjunction with the highbinding efficiency of the ligand.

In some embodiments, the high binding specificity and/or affinity ofcapture support 204 to the target component may be because the onlyknown interaction of the capture ligand is exclusive to sTNF-R1 and/orsTNF-R2 in the plasma. This binding specificity may result from the useof the sc-TNF ligand described above, the trimeric form of the sc-TNFligand, the materials used to form (e.g., the chemical composition of)the beads and/or the specific bead matrix, use of ethanolamine to quenchthe beads (e.g., used to reduce non-specific binding), optimization oftarget protein binding versus non-specific binding, the pore size usedfor filters 212 and 214, and/or other factors.

In some embodiments, a binding affinity of the capture support to the atleast one target component is at least about 10⁻⁵ K_(D) or greater. Insome embodiments, a binding affinity of the capture support to the atleast one target component is at least about 10⁻⁶ K_(D). In someembodiments, a binding affinity of the capture support to the at leastone target component is at least about 10⁻⁷ K_(D). In some embodiments,a binding affinity of the capture support to the at least one targetcomponent is at least about 10⁻⁸ K_(D). In some embodiments, a bindingaffinity of the capture support to the at least one target component isat least about 10⁻⁹ K_(D). In some embodiments, a binding affinity ofthe capture support to the at least one target component is at leastabout 10⁻¹⁰ K_(D). In some embodiments, a binding affinity of thecapture support to the at least one target component is at least about10⁻¹¹ K_(D). In some embodiments, a binding affinity of the capturesupport to the at least one target component is at least about 10⁻¹²K_(D). In some embodiments, a binding affinity of the capture support tothe at least one target component is at least about 10⁻¹³ K_(D). In someembodiments, a binding affinity of the capture support to the at leastone target component is at least about 10⁻¹⁴ K_(D).

In some embodiments, system 10 may be configured such that it has aleach rate of TNF less than 1/1000^(th) of maximum tolerable daily dose(MTD) limits (see, for example, Goossens, V., et al. (1995) Proc. NatlAcad. Sci. USA, 92, 8115-8119). In some embodiments, system 10 may beconfigured such that it has a leach rate less than 1/10000th of MTD doselimits. In some embodiments, system 10 may be configured such that ithas a leach rate less than 1/500th of MTD daily limits. In someembodiments, system 10 may be configured such that it has a leach rateless than 1/100th of MTD daily limits. This may ensure the clinicaleffectiveness of system 10 including successful, efficient, and specificcapture of the one or more target components, unbiased by clinicaleffects and/or side effects resulting from escape (i.e., leaching) ofportions of capture support 204 (e.g., the ligands described herein)into the patient's circulatory system.

Leach rate may be defined as the percent of capture support 204 thatescapes system 10 (e.g., in units of ng/mL/min) relative to the totalvolume of capture support 204 contained in system 10's sequesteringchamber 202 following production. For example, in some embodiments,system 10 may have a leach rate of less than about 150 ng/mL/min. Insome embodiments, system 10 may have a leach rate of less than about 100ng/mL/min. In some embodiments, system 10 may have a leach rate of lessthan about 80 ng/mL/min. In some embodiments, system 10 may have a leachrate of less than about 50 ng/mL/min. In some embodiments, system 10 mayhave a leach rate of less than about 40 ng/mL/min. In some embodiments,system 10 may have a leach rate of less than about 30 ng/mL/min. In someembodiments, system 10 may have a leach rate of less than about 20ng/mL/min. In some embodiments, system 10 may have a leach rate of lessthan about 10 ng/mL/min.

The leach rate may result from the strength and integrity of the (e.g.,chemical and/or electrostatic) bonding between the sc-TNF ligands andthe beads, the use of reductive amination chemistry with a specificligand, the use of the trimeric form of the sc-TNF ligand, the (e.g.,chemistry and/or electrostatic) process used for coupling the ligands tothe beads, the clinical pretreatment approach for preparing system 10for patient treatment (e.g., volume and flow-rate of pre-use flushing),a clinically-practical flow-rate through system 10 that balances captureefficiency versus clinical procedure time, cleaning housing 16 duringmanufacturing, the sterilization technique and radiation dosage utilizedin production, and/or other factors. In some embodiments, the clinicalpretreatment preparation of system 10 (e.g., volume and flow-rate ofpre-use flushing) comprises a one-liter flush of normal saline at a flowrate of about 100 mL/min.

By way of a non-limiting example, FIG. 4 and FIG. 5 illustrate capturesupport 204 (e.g., beads 400 in this example) in sequestering chamber202 of housing 16 binding to a target component 402 (sTNF-R1) and/or asecond target component 403 sTNF-R2 (these are just examples—more targetcomponents are possible) of body fluid (plasma 404 (comprised of sTNF-R1402 and sTNF-R2 403) in this example) to capture target component 402and 403 within the sequestering chamber 202. FIG. 5 is an enlarged viewof a bead 400 (e.g., a portion of the capture support) shown in FIG. 4.FIG. 5 shows the direction 500 of body fluid (e.g., plasma 404) flowthrough system 10 (FIG. 1-3) from inlet 200 to outlet 210 (FIG. 1-3). Insome embodiments, system 10 may be symmetrical and may be bidirectionalsuch that a clinical user could select to use either end of theapparatus to serve as the upstream inlet or downstream outlet.

As shown in FIG. 4 and FIG. 5, the capturing occurs when there iscontact between capture support 204 and the body fluid (plasma 404).Specifically, the capturing occurs responsive to close proximity and/ordirect contact between the sTNF-R1 and sTNF-R2 molecules 402, 403 inplasma 404 and ligands (e.g., the sc-TNF ligands described above) 410that are bound (coupled) to beads 400. Bonds 412 between ligands 410 andbeads 400 are also shown. Capture support 204 (e.g., the combination ofbeads 400 and ligands 410) may be configured to bind to one or moretarget components 402, 403 (sTNF-R1 and R2 molecules) to reduce anamount of one or more target components 402, 403 in the body fluid(e.g., plasma 404).

In some embodiments, once the treated sTNF-R deficient plasma passesthrough outlet 210 (FIG. 2 and FIG. 3), some or all of the treatedsTNF-R deficient sample may be reinfused into the patient (e.g., via theapheresis machine shown in FIG. 1). As sTNF-Rs are removed, thereservoir of uncomplexed sTNF-Rs in the patient's blood is reduced,resulting in a concentration equilibrium shift toward increasedavailability of TNF to promote anti-cancer effects at the tumor site(s).

As described herein, system 10 (FIG. 1-3) may be configured toselectively remove immune suppressive factors associated with states ofimmune suppression. In some embodiments, the immune suppressive factorsare soluble TNF receptor 1 and soluble TNF receptor 2 (sTNF-Rs).Removing sTNF-Rs from the patient's blood, plasma, and/or other bodyfluid may result in increased availability of endogenous TNF, whichpromotes anti-cancer effects at tumor site(s).

Returning to FIG. 2 and FIG. 3, access ports 206, 208 may be configuredto provide access to sequestering chamber 202. This access may beseparate from access via inlet 200, outlet 210, and/or other accesspoints. Access ports 206 and 208 may be configured to facilitateinsertion and/or removal of capture support 204 to or from sequesteringchamber 202. This may be performed during manufacturing of system 10,for example, and/or at other times.

In some embodiments, capture support 204 may be suspended in apreservative buffer solution for storage prior to use in system 10. Insome embodiments, the preservative buffer solution comprisesbacteriostatic saline, bacteriostatic phosphate, and/or other solutions.In some embodiments, the preservative buffer solution may bebacteriostatic phosphate buffered saline (PBS) that may contain 0.9%benzyl alcohol. Capture support 204 may be refrigerated in solution at2-8° C. until ready for subsequent aseptic filling of housing 16. Insome embodiments, system 10 may be stored at 2-8° C. between its time ofmanufacture, shipping, and/or clinical use.

In some embodiments, the preservative buffer solution is flushed fromsystem 10 (e.g., via inlet 200 and outlet 210) immediately prior toclinical use of system 10. In some embodiments, access ports 206, 208comprise Luer fittings having caps and/or other components. In someembodiments, access ports 206 and/or 208 have corresponding caps and/orother components. The corresponding caps may be formed frompolycarbonate and/or other materials.

FIG. 6 illustrates an example regeneration mechanism 600 configured tocouple with system 10. In some embodiments, regeneration mechanism maybe considered to be an additional part of, and/or an extension of system10. As shown in FIG. 6, mechanism 600 may include a rinsing solution 602source, a regeneration solution 604 source, a pump 606, a waste line608, and/or other components. In some embodiments, rinsing solution 602and regeneration solution 604 may be alternately pumped through system10 by pump 606 and into waste line 608.

In some embodiments, system 10 may include a target component outletport configured to facilitate sampling or removal of all or part of thecaptured target component (e.g., without compromising the extracorporealclosed-circuit column formed by system 10).

In some embodiments, system 10 may be configured to facilitate reuse byalternating capture and dissociation steps. Dissociating the targetprior to further capture may regenerate the column to about the originalspecifications and function. To accomplish this dissociating, thecaptured complexes between bead-bound proteins and their ligands isdisrupted, resulting in a dissociated complex. Such a dissociation stagemay be application dependent so the particular dissociation conditionsmay depend upon the particular subtype of circulating protein orcomplexed moiety that was captured. Agents that cause high saltconcentrations, such as chaotropic agents or low pH may be effectivedissociation agents. In order to dissociate the capture complex highsalt, such as sodium chloride (300 mM-1,5M) or a chaotropic agent suchas guanidine hydrochloride 3-8M, concentrations may be used. In someembodiments, a salt solution may have a pH of approximately 7.2 andcomprise either 500 mM NaOH, 2 mM EDTA and 50 mM Tris buffer, or 500 mMNaOH, 2 mM EDTA and 50 mM sodium phosphate. Alternatively, the capturedimmune complexes may be dissociated with a low pH solution. Theeffective pH for the dissociation stage may be approximately 2.8, forexample. An example pH range may be 1.5 to 2.5. This may be accomplishedwith pH adjusted citrate or glycine solutions. In some embodiments, thepH range may be 2.0 to 2.5. In some embodiments, the pH range may beabout 2.5-3.5. However, it should be realized that the lower the pH, theshorter the dissociation time needed. Acids, such as acetic acid, citricacid and hydrochloric acid, for example, may be used for lowering thepH. In addition to either raising the salt concentration or lowering thepH, other methods of dissociating immune complexes are also possible. Inaddition to either raising the salt concentration or lowering the pH,the dissociation conditions may be configured to occur for a shortperiod of time and include bovine serum albumin (BSA) and/or otherligand or receptor competing components.

In some embodiments, system 10 may include an elution reagent portconfigured to facilitate introduction of an elution reagent into thesequestering chamber (e.g., without compromising the extracorporealclosed-circuit column formed by system 10). The elution reagent port maybe further configured to receive a conditioning agent configured toprepare system 10 for reuse. In some embodiments, the elution reagentport(s) may be the same as one or both of access ports 206 and/or 208.

In some embodiments, pump 606 may be configured to drive theelution/regeneration agents through inlet 200 (FIG. 2), sequesteringchamber 202 (FIG. 2), and outlet 210 (FIG. 2). Pump 606 may comprise asyringe pump, a peristaltic pump, a piston pump, a diaphragm pump, acombination thereof, and/or other pumps.

In some embodiments, system 10 may comprise one or more additionalsequestering chambers 202, as depicted in FIG. 7. FIG. 7 illustrates twoexample embodiments 700 and 750 of system 10 having the additionalsequestering chambers 202. In example embodiment 700, sequesteringchambers 202A and 202B are positioned within housing 16 in series. Inexample embodiment 750, chambers 202C, 202D, and 202E are positionedwithin housing 16 in parallel. The different chambers (e.g., 202A and B,and 202 C-E) may be separated by filters 212, 214 (and/or otherfilters), separating membranes 710, and/or other components. In someembodiments, separating membranes 710 may be filters 212 and/or 214 andvice versa, for example.

These additional sequestering chambers 202A-E may include capturesupports similar to and/or the same as capture support 204 (FIG. 2)described above, or different capture supports. The additionalsequestering chambers 202 (A-E) and capture supports may be configuredto function similar to and/or the same as sequestering chamber 202 andcapture support 204. In some embodiments, the one or more additionalsequestering chambers (202A-E) may be combined (e.g., 202A and B, and202C, D, and E) to form a multistage separation circuit configured tobind with a plurality of different target components. In someembodiments, the multiple sequestering chambers may be configured in aserial configuration (e.g., embodiment 700) where the full volume ofbody fluid (plasma, for example) flowing through system 10 passessequentially through each of the plurality of sequestering chambers(e.g., 202A then 202B). In some embodiments, the multiple sequesteringchambers may be configured in a parallel configuration (e.g., embodiment750) where the volume of body fluid (plasma, for example) flowingthrough system 10 is distributed into equal or nonequal fractions suchthat these fractionated sub-volumes of fluid are passed in parallelthrough each of the plurality of sequestering chambers (e.g., 202C,202D, and 202E).

FIG. 8 illustrates additional embodiments 800 and 850 where a pluralityof systems (e.g., a plurality of system 10's) may be configured into asingle plasmapheresis flow circuit 802 or 852 at the same time during atreatment procedure. In one embodiment (e.g., 800), the plurality ofsystems (10) may be cascaded in series where, plasma for example, flowsthrough one system 10 and then next. In another embodiment (e.g., 850),two or more systems (10) may be configured in parallel where the volumeof body fluid (plasma, for example) flowing through the plasmapheresisflow circuit may be distributed into equal or nonequal fractions suchthat these fractionated sub-volumes of fluid may be passed in parallelthrough each of the plurality of systems (e.g., two systems 10 are shownin embodiment 850 but this is not intended to be limiting). In oneembodiment involving such a parallel system configuration, the totalvolume of fluid in the plasmapheresis flow circuit may be initiallydirected to pass through one or more of the parallel system sequesteringchambers (e.g., 202 as described above), and then at a subsequent timeduring the same treatment procedure, fluid in the circuit may beredirected to pass through a different system or subdivided to passthrough a different combination of the parallel systems coupled withinthe flow circuit. In any of the configurations described above, eachindividual system may contain the same or different capture matricestargeted at the same or different target agents in the body fluid.

FIG. 9 illustrates an embodiment 900 of system 10 comprising fluid inlet200 for the fluid to enter system 10, multiple sequestering chambers902, 904, 906, and 908 in parallel, each with an independent flow path(903-909) from system 10 inlet 200 to outlet 210 (which may be formed bymale Luer ports for example), and each with its own flow control valve910, 912, 914, 916 (e.g., stopcocks and/or other components) allowing anindividual sequestering chamber to be turned “on” or “off”. In thisembodiment, fluid can be directed through any one chamber or anycombination of multiple chambers at the same time. An example flow path950 is shown in the bottom portion of FIG. 9. Fluid existing the one ormore chambers being used at a time, is recombined at the system fluidoutlet 210 for return to the apheresis machine. Within this embodiment,each sequestering chamber 902-908 can include one or more differentcapture molecules (e.g., included in capture support 204 describedabove) configured to target one or more different target portions in thebody fluid. As shown in FIG. 10, the plurality of sequestering chambers902-908 and their corresponding flow control valves 910-916 may beincluded within a single, unitary outer housing (e.g., 16 as describedabove).

FIG. 10 illustrates method 1000 for removing the target component of theblood, plasma, and/or other body fluid with system 10 (FIG. 1-3). Theoperations of method 1000 presented below are intended to beillustrative. In some embodiments, method 1000 may be accomplished withone or more additional operations not described, and/or without one ormore of the operations discussed. Additionally, the order in which theoperations of method 1000 are illustrated in FIG. 10 and described belowis not intended to be limiting.

At an operation 1002, blood, plasma, and/or other body fluid may beconducted from a patient (e.g., patient 14 shown in FIG. 1) throughinlet 200 (FIG. 2-3) to sequestering chamber 202 (FIG. 2-3).

At an operation 1004, the target component of the blood, plasma, and/orother body fluid may be bound to capture the target component insequestering chamber 202 (FIG. 2-3) to reduce the amount of the targetcomponent in the blood, plasma, and/or other body fluid.

At an operation 1006, the body fluid having the reduced amount of thetarget component is passed from sequestering chamber 202 (FIG. 2-3)through outlet 210 (FIG. 2-3) for reintroduction back into the patient.

At an operation 1008, the reduced amount of the target component in thebody fluid reintroduced back into the patient where it may or may not bequantitatively measured (e.g., operation 1008 may be optional). In someembodiments, where quantitative measuring is used, the measuring maycomprise one or more of performing liquid chromatography-massspectrometry (LC-MS), high performance liquid chromatography (HPLC),ultra-high performance liquid chromatography (UHPLC), resistancemeasurements, light emission measurements, chemiluminescence,electroluminescence, electrochemiluminescence, chromatographicmonitoring, positron emission tomography (PET), x-ray computedtomography (CT), magnetic resonance imaging (MRI), ultrasound, gammacamera, single photon emission computed tomography (SPECT), an enzymelinked immunosorbent assay (ELISA), surface plasmon resonance (SPR)measurements, and/or other operations.

At an operation 1010, a leach rate of the capture support in the bodyfluid reintroduced back into the patient may or may not be measured(e.g., operation 1010 may be optional). The concentration of the captureagent (TNF, for example) may be determined from fluid samples drawn fromthe inlet 200 and the outlet 210 and/or from fluid connectors attachedthereto. The difference between the two measured concentration valuesmay be used to determine the rate and amount of capture agent escapingthe sequestering chamber. In some embodiments, flow rate in mL/min maybe utilized to determine the total amount of capture agent leached fromthe system and into the patient's circulatory system over a period oftime (the duration of a single treatment procedure, for example).

Although the system(s) or method(s) of this disclosure have beendescribed in detail for the purpose of illustration based on what iscurrently considered to be the most practical and preferredimplementations, it is to be understood that such detail is solely forthat purpose and that the disclosure is not limited to the disclosedimplementations, but, on the contrary, is intended to covermodifications and equivalent arrangements that are within the spirit andscope of the appended claims. For example, it is to be understood thatthe present disclosure contemplates that, to the extent possible, one ormore features of any implementation can be combined with one or morefeatures of any other implementation.

The following appended examples provide various demonstrations ofsuccessful use of various embodiments of the systems and methodsdescribed herein.

Example 1—Materials and Assembly of the Present System

MATERIALS—The immobilized solid support matrix (capture support 204)utilized in this embodiment of the system was an agarose-based bead(i.e., a strong crosslinked support resin).

CHEMISTRIES—The solid support matrix (capture support 204) was activatedusing sodium metaperiodate, which was then coupled to a single chain TNFligand by reductive amination. The amount of 1 mg of TNF was coupled permL of solid support matrix.

ASSEMBLY—The binding matrix (capture support 204) includes asingle-chain TNF (SC-TNF) protein that is covalently linked to agarosebeads. The device housing is comprised of a polycarbonate tube with twoside ports for filling, capped with non-vented Luer caps. The end capshave male Luer ports and are tightly bound to the tube to create afluidically sealed enclosure terminated at each end by the end cap Luerports. Internal to the housing at the juncture between each end cap andthe tube is a filter frit with an average pore size of 15-50 μm toretain the TNF ligand-coupled beads, the minimum diameter of whichexceeds the pore size of the filter (e.g., as described above).

Example 2—Parameters for the Utilizing the Present System and Method

1. In configurations where pre- and post-column plasma concentrationmeasurements are being taken to determine capture efficiency and/orleaching, which is not necessary for routine clinical applications,3-way stopcock valves are attached to the inlet and outlet of the columnend caps to enable periodic plasma sampling. 2. The system along withthe attached stopcocks are connected into the plasma circuit of theapheresis machine. 3. The apheresis unit is configured withprocedure-independent and/or procedure-specific operating parameters perthe apheresis machine's instructions for use to complete steps 4, 6, and10 below. 4. Flush the system with 1 L of normal saline prior toconnecting the patient to the apheresis machine. 5. Connect the patientto the apheresis machine. 6. Treat the patient with the present system.7. If applicable, collect blood and plasma samples in accordance with astudy protocol and/or clinical treatment procedural plan. 8.Continuously monitor the patient's vital signs in accordance withtypical apheresis treatment practice. 9. Continuously monitor thepatient for adverse events before, during, and after the treatment. 10.Flush/rinse the device with normal saline post-treatment, re-cap thecolumn with the retained end caps, and ensure proper storage and/ordisposal of the used device.

The column of the present invention is intended to be used inconjunction with apheresis machines, such as the Terumo BCT or SpectraOptia System, for example, that are designed to accommodate plasmaprocessing columns such as system 10. Such systems automate calculationsbased on the patient data and device parameters that are configured bythe operator. In the event that the apheresis machine does not automatesuch calculations, the formulas identified in the tables below may beused.

TABLE 1 Formula for Calculating Total Blood Volume Metric Female TBV =183 + (356 × H³) + TBV = total blood Units (33.1 × W) volume (mL) MaleTBV = 604 + (367 × H³) + H = height (m) (32.2 × W) W = weight (kg)English Female TBV = 183 + (0.005835 × H³) + TBV = total blood Units (15× W) volume (mL) Male TBV = 604 + (0.006012 × H³) + H = height (in)(14.6 × W) W = weight (lbs)

TABLE 2 Formula for Calculating Patient Plasma Volume Vp = 0.065 × W ×(1 − HCT) Vp = patient plasma volume (mL) W = weight (kg) HCT =hematocrit (%)

TABLE 3 Formula for Calculating Treatment Time T = (Vp × Vx)/Q T =Treatment time (min) Vp = Patient plasma volume (mL) Vx = # of plasmavolumes to be treated (unitless) Q = Flow rate (mL/min)

INTENDED CLINICAL PERFORMANCE—The immunopheresis column of (e.g., system10) is a device designed to successfully integrate with commerciallyavailable apheresis machines that use centrifugal or membrane separationtechniques and that allow for secondary plasma processing (e.g., such asby system 10) to efficiently remove sTNF-Rs from the patient's plasma.The system disclosed herein meets the clinical performance requirementsdescribed below. Specified treatment times and flow rates have beenverified to fall within the capabilities of modern apheresis systemsusing centrifugal separation techniques.

System Performance Specifications—1. Biological safety: Demonstrated tobe biologically safe for use as an extracorporeal device supportingprolonged exposure to circulating blood. 2. Binding target: sTNF-Rs(includes sTNF-R1 and sTNF-R2). 3. Binding efficiency: >80% following 30minutes of treatment at clinically-relevant flow rates. 4. Bindingcapacity: >230 pg of sTNF-Rs. 5. Flow rate (plasma): 60 mL/min. 6.Treatment time: approximately 2 hours. 7. Target plasma exchangevolumes: 2 plasma volumes. 8. Leaching rate (TNFα): <50 ng/min. 9. Shelflife: >6 months @ 2-8° C. 10. Pressure tolerance: 776 mmHg.

Example 3—Efficacy of System 10 Based on In Vitro Parameters

PRECLINICAL TESTING—In vitro testing demonstrated that system 10 meetsthe performance and safety specifications defined in “Intended ClinicalPerformance” (previous section). The TNF-coupled binding matrixeffectively captured over 98% of the sTNF-Rs in both spiked buffersolution and human plasma in the standard in vitro test, whilemaintaining a calculated sc-TNFα leaching rate of <50 ng/min (equivalentto a maximum of 6 pg in 2 hours), which is well below acceptable safetylimits since the maximum tolerated dose (MTD) per day of TNFα isapproximately 200 pg (see, for example, Goossens, V., et al. (1995)Proc. Natl Acad. Sci. USA, 92, 8115-8119). These performance and safetymeasures were replicated after beads were sterilized by E-Beamirradiation (15-30 kGy). Additionally, in this example, system 10 isdesigned for sustained flow rates of <300 mL/min, between 10 and 44mL/min, and/or at 45 to 100 mL/min. The housing (e.g., 16 shown in FIG.2 and FIG. 3) can withstand internal pressures of >776 mmHg, which iswell above the typical back-pressure shutoff thresholds for commonlyused apheresis machines.

Characterization of Binding Matrix (e.g., capture support 204)—Identity,purity, and integrity of the TNF ligand—A recombinant single chain TNFαligand (sc-TNFα ligand, comprising 3 TNFα monomers) was used as abinding agent to capture sTNF-Rs. The TNFα has been characterized byassessing purity by high performance liquid chromatography (HPLC),integrity and identity by mass spectrometry, and functional strength bya flow test binding assay. HPLC was performed using a column with aphosphate buffer as the mobile phase at a flow rate of 1 mL/min. Theintegrity was measured by mass spectroscopy, which confirmed thecalculated molecular weight of ˜54 kD.

Binding efficiency of the binding matrix in sTNF-R spiked buffer—For thestandard flow test, the amount of 2 mL of bead bed was obtained from anirradiated column of the present invention and transferred to a smallcolumn and tested for capture efficiency from phosphate buffer spikedwith sTNF-R1 or sTNF-R2. TNF-R1 spiked buffer (20 ng/mL) was passedthrough the column at 10 ml/min, 5 mL/min/, 2.5 mL/min, 5 mL/min and 10mL/min, respectively. Samples were collected at 0.5 min intervals andassayed for sTNF-Rs. The result was greater than 98% capture efficiencyfor all the samples. Assaying for TNFα showed its release to be lessthan 7 ng/min.

Capture capacity of the binding matrix in sTNF-R spiked human plasma—Totest the capacity of system 10, a column of the present invention wastested by running 1.8 liters of spiked human plasma through the deviceat flow rate of 45 mL/min. The human plasma used for each run was spikedwith high concentrations of each receptor (9.74 ng/mL for sTNF-R1 and127.5 ng/mL sTNF-R2). Samples of the post-column plasma were collectedevery 10 minutes and analyzed for sTNF-R concentration using acommercially-available, high-precision sTNF-R1/sTNF-R2 diagnostics kit.The amount of sTNF-R retained on the column was calculated to verify thecapture efficiency and overall capacity.

Binding efficiency at each time point was calculated by comparing thepre- and post-column concentrations of sTNF-R. The binding matrixcaptured greater than 85% of sTNF-R1 and sTNF-R2. The binding efficiencywas greater than 95% for sTNF-R1 throughout and although a lesserefficiency was observed for sTNF-R2, its capture still remaining atgreater than 85%. The slight linear decrease in binding efficiency forsTNF-R2 is possibly attributed to the lower binding affinity of thissoluble receptor for the TNFα ligand used.

The total amount of sTNF-R captured on the columns was determined bycomparing pre- and post-treatment concentrations of sTNF-Rs. The columncaptured 230 μg of sTNF-R in the 15 mL/min run and 149.4 μg in the 45mL/min run. Both of these values are in excess of the amount ofapproximately 30 μg of sTNF-Rs that is typically present in a cancerpatient. The amount of total TNF-R capture for the 15 ml/min run at 80minutes, the point where the column efficiency began to decrease, was99.9 μg, which is still well in excess of the typical amount present ina cancer patient.

Leaching rate of TNFα from the device of the present invention—System 10is configured to prevent the unintentional release (i.e., ‘leaching’) ofTNFα from system 10 during its use to avoid infusing potentiallypharmacologically-significant amounts into the patient. The TNF releasefrom the beads was determined during conduct of a flow test. Phosphatebuffered saline was spiked with approximately 20 ng/mL of sTNF-R1 andrun through a 2 mL bead bed obtained from a system 10 (FIG. 1-3) column.The flow rate was sequentially performed at 10 mL/min, 5 mL/min, 2.5ml/min, 5 mL/min and 10 mL/min for 30 seconds at each flow rate. Sampleswere analyzed according to a commercially-available, high-precision TNFdiagnostics kit. The TNF leaching rate in ng/min was calculated bymultiplying the concentration of TNF in the sample (ng/mL) by the flowrate (mL/min). The amount of release in the final fraction collected at10 mL/min was 0.65 ng/min per 2 mL of beads or 0.325 ng/min/mL (0.65ng/min/2 mL). In this example, the upper limit of the bead bed volumethat would be within the specification of 50 ng/min is (50ng/min)/(0.325 ng/min/mL) or 154 mL of beads.

Device housing integrity—Testing was conducted on empty housings (e.g.,16) of the present invention to verify that the integrity of system 10is maintained at increased internal pressures. A side port (e.g., 206,208) of system 10 was attached to a compressor using tubing and aconnector. The capped housing was submerged in a water bath, pressurizedto 776 mmHg and observed for air bubble generation. No device failures(presence of bubbles leaking from the submerged device) were observed.This test pressure exceeds the maximum pressure of the Optia unit (500mmHg).

Example 4—Demonstration of Efficacy in a Canine Model

The effect of extracorporeal removal of sTNF-Rs, with system 10, fromcanines with naturally occurring solid malignant tumor or melanoma(Stage 4) was assessed in a proof-of-concept comparative oncology study.The study used system 10 with a sc-TNFα peptide-bead matrix. System 10was used in conjunction with the Terumo BCT Spectra Optia ApheresisSystem for secondary plasma processing through system 10. Dual lumencatheters were employed in most dogs for vascular access, andextracorporeal anticoagulation was achieved with acid-citrate dextroseanticoagulation (ACDA) solution and reversed with calcium gluconateinfusion as recommended per the Spectra Optia user operating manual. Atotal of 20 canines were treated. Canine patients received between 12-24apheresis treatments over the course of the 4-8 week treatment phase ofthe study. Over 300 immunopheresis treatments were performed during thisstudy.

Device Performance—To verify the in vivo safety and performance of thedevice, TNF and sTNF-Rs were measured every 30 minutes during treatmentin pre- and post-column plasma (taken from the inlet and outlet ports ofthe device, respectively); and systemic (from blood) TNF and sTNF-Rswere measured before each treatment, every 30 minutes during treatment,and every 30 minutes after treatment (for the 1.5 hours immediatelyfollowing treatment).

During each treatment, analysis of pre- and post-column (system 10)plasma showed that sTNF-R was efficiently and consistently removed overthe course of each individual treatment without a measurable increase inTNF levels in the blood. Analysis of pre- and post-treatment plasmagenerally showed nearly a 50% reduction in systemic sTNF-R levels.

Device Safety and Clinical Efficacy—To evaluate the clinical safety andefficacy of the device, measurements of safety, tolerability, and impacton tumor progression were made throughout the study.

During the course of over 300 administered immunopheresis treatmentsacross a total of 20 canines, there were few serious adverse eventsreported, and none that were attributed to the specific apheresisprocedure or as a result of treatment with system 10 or removal ofsTNF-Rs. The tolerability to treatment is best illustrated whenexamining the canine patient QoL data. Changes in QoL were generallyscored as ‘neutral’ for most scores throughout the active treatmentphase of the study, which included between 12-24 treatments per patient.Although some scores worsened, most showed stable parameters orimprovement throughout the entire course of treatment, which for theStage 4 patient population in the study represents a favorable outcome.

Treatment with the device of the present invention had an overallbeneficial effect on tumor progression. The majority of canines (12/17evaluable cases) were scored as “stable disease” (SD) at some pointduring treatment (data not shown) and 7 of the 17 evaluable caninesshowed a favorable treatment-related effect at the end of the treatmentphase, with one case showing complete regression (CR).

Conclusions—The overall condition of the canines generally improvedwhile they were on study, with an observable stabilization or reductionin tumor burden and an improvement in quality of life. In addition, theabsence of clinically-significant safety issues are consistent with theestablished relative safety of general apheresis procedures and iscompelling, as it presents the potential to provideclinically-meaningful benefit without the typical and significant sideeffects associated with traditional chemotherapy and radiation. Thiscanine companion animal study showed that extracorporeal removal ofsTNF-Rs utilizing the Apheresis Immunoadsorption Affinity Column of thepresent invention containing a sc-TNF peptide-bead matrix could betherapeutically effective in canines with cancer and provided compellingclinical evidence that use of such a device could be employed in humansubjects.

Example 5—Biocompatibility

System 10 has been evaluated for biological safety in accordance withthe Food & Drug Administration (FDA) Biocompatibility Testing Matrix andInternational Standard ISO 10993-1 (2009). System 10 can be categorizedas an External Communicating Device, Circulating blood, with prolongedcontact duration. This is the same testing categorization utilized formultiple other commercially available extracorporeal immunosorbentcolumns.

Biocompatibility tests were performed in accordance with 21 CFR Part 58(Good Laboratory Practice for Nonclinical Laboratory Studies). All testswere conducted on the sterilized, finished devices by a certified,independent testing organization. Based upon the tests performed, thedevices conform to the recommendations and principles contained withinthe ISO 10993-1 (2012) consensus standard, “Biological evaluation ofmedical devices—Part 1: Evaluation and testing within a risk managementprocess,” and with the FDA's associated guidance document issued Jun.16, 2016.

Example 6—Demonstration of Safety in Human Subjects

A first-in-man, compassionate use clinical study has been conducted tocollect pilot safety and performance data of system 10. The device wasused in combination with the Terumo BCT Spectra Optia System withsecondary plasma processing through system 10 as was done in thecompanion canine comparative oncology study (described above). Duallumen catheters were employed for vascular access, based on experiencefrom the canine study, and extracorporeal anticoagulation (ACDAsolution) and reversal (with calcium gluconate infusion) was similar tothat employed in the canine study and as recommended per the SpectraOptia user operating manual.

Apheresis Procedure Performance—System 10 was successfully utilized withthe Terumo BCT Spectra Optia's apheresis equipment. For each treatmentconducted, system 10 was able to be appropriately integrated into theextracorporeal plasma circuit. No device-attributed obstructions in thesecondary plasma processing circuit (e.g., the circuit coupled to system10) were reported. The system circuit integrity was consistentlymaintained (e.g., no leaks/fluid losses were reported) and alltreatments were able to be successfully completed. Based upon thiscollective data, the device of the present invention appears to besuitable for use with the Terumo BCT Spectra Optia.

Safety and Tolerability Results—A total of 14 patients were enrolled inthe study. All patients had advanced cancer for which current treatmentshad failed but were otherwise stable (baseline Eastern CooperativeOncology Group (ECOG) score 0-2). The range of treatments each patientreceived varied (e.g., one patient received up to 16 treatments). Atotal of 93 individual treatments were completed with no unanticipatedapheresis-related adverse events (AE) or adverse device effects (ADE)being reported. Based on the data collected from this study,Immunopheresis using the present approach and system 10 appearsgenerally safe and well-tolerated.

FIG. 11 illustrates representative system 10 (FIG. 1-3) performancecharacteristics including sTNF-R1 and sTNF-R2 reduction from a humanpatient's blood pool and column capture efficiency as a function ofprocedure time. FIG. 11 is illustrative of typical system 10 performanceresults from a single human patient treatment. Samples of whole bloodand plasma where drawn at baseline (T=0 mins), 30 mins., 60 mins., 90mins., and at the end of treatment (i.e., completion of 2 plasma volumescirculated through the system), which in this example occurred 175minutes from the procedure start time. At each time point, whole bloodwas drawn from the patient's central line catheter and plasma sampleswere taken at the inlet (e.g., 200 shown in FIGS. 2-3) and outlet (e.g.,210 shown in FIGS. 2-3) of system 10. Additionally, whole blood sampleswere drawn 30- and 60-minutes post-treatment. sTNF-R1 and sTNF-R2concentrations in the samples were analyzed using acommercially-available, high-precision diagnostics assay. FIG. 11 showsa significant differential between corresponding inlet 200 and outlet210 sTNF-R1 and sTNF-R2 concentrations at each time point demonstratingthat system 10 was effectively capturing sTNF-R1 and sTNF-R2 throughoutthe course of treatment. Moreover, the steady time-based reduction insTNF-R1 and sTNF-R2 concentrations observed in the patient's overallcirculatory system (i.e. central line whole blood measurements) followedby rebounding levels 30- and 60-minutes post-treatment, indicates thetherapeutic objective of reducing endogenous levels of sTNF-R1 andsTNF-R2 during the treatment period was effectively being accomplished.

1-41. (canceled)
 42. A system for removing a target component from abody fluid of a patient, the system comprising: a housing; an inletcoupled to the housing and configured to receive the body fluid; aplurality of sequestering chambers disposed within the housing andconfigured to each receive a portion of the body fluid from the inlet;and an access port coupled to the housing and configured to facilitateinsertion and/or removal of a capture support to or from one of theplurality of sequestering chambers.
 43. The system of claim 42, whereinthe access port comprises a plurality of access ports respectivelyconfigured to facilitate insertion and/or removal of one of a pluralityof capture supports to or from one of the plurality of sequesteringchambers.
 44. The system of claim 42, wherein the plurality ofsequesting chambers are arranged in parallel, with each sequestingchamber coupled independently to the inlet.
 45. The system of claim 42,wherein: the plurality of sequesting chambers are arranged in series; afirst sequesting chamber is coupled to the inlet and configured toreceive all of the body fluid from the inlet; and the first sequestingchamber is coupled to a second sequesting chamber such that all of thebody fluid passes from the first sequesting chamber to the secondsequestering chamber.
 46. The system of claim 45, wherein the secondsequesting chamber is coupled directly to the outlet such that all ofthe body fluid passes from the second sequestering chamber to theoutlet.
 47. The system of claim 42, further comprising an outlet coupledto the housing and configured to receive the respective portions of thebody fluid from the plurality of sequestering chambers.
 48. The systemof claim 47, further comprising a plurality of filters disposed withinthe housing so as to separate the plurality of sequestering chambersfrom the inlet and the outlet.
 49. The system of claim 42, furthercomprising a capture support disposed within one of the plurality ofsequestering chambers and configured to bind to a target component. 50.The system of claim 49, wherein the capture support comprises aplurality of capture supports respectively disposed within the pluralityof sequestering chambers and respectively configured to bind to one of aplurality of target components.
 51. The system of claim 49, wherein: thecapture support comprises a portion of a tumor necrosis factor (TNF);and the target component comprises a soluble TNF receptor.
 52. Thesystem of claim 51, wherein the capture agent comprises a single chainTNF.
 53. The system of claim 52, wherein the single chain TNF comprisesat least three TNF monomers or portions thereof.
 54. The system of claim42, wherein the capture support comprises a solid support.
 55. Thesystem of claim 54, wherein the capture support comprises ligands boundto beads.